Structure and Dynamics of Supramolecular Polymers: Wait and See

The introduction of stereogenic centers in supramolecular building blocks is used to unveil subtle changes in supramolecular structure and dynamics over time. Three stereogenic centers based on deuterium atoms were introduced in the side chains of a benzene-1,3,5-tricarboxamide (BTA) resulting in a supramolecular polymer in water that at first glance has a structure and dynamics identical to its achiral counterpart. Using three different techniques, the properties of the double helical polymers are compared after 1 day and 4 weeks. An increase in helical preference is observed over time as well as a decrease in the helical pitch and monomer exchange dynamics. It is proposed that the polymer of the chiral monomer needs time to arrive at its maximal preference in helical bias. These results indicate that the order and tight packing increase over time, while the dynamics of this supramolecular polymer decrease over time, an effect that is typically overlooked but unveiled by the isotopic chirality.

Fourier-Transform infrared (FT-IR) spectra were recorded on a Perkin Elmer Spectrum Two FT-IR spectrometer. Solid state samples were measured at room temperature from 4000 cm -1 to 450 cm -1 over 16 scans. Liquid FT-IR measurements were performed using a CaF2 Liquid Cell with an optical path length of 0.05 mm. Samples for FT-IR in solution were prepared at a concentration of 20 mg/mL to facilitate dissolution. BTA material was weighed and added to a clean vial. All samples were dried overnight with approximately 5 grams of P2O5 in a separate beaker in the vacuum oven at 40 °C. Sample in MeOD were prepared by adding MeOD to the vials to obtain the desired concentration. The sample in D2O was prepared by addition of the solvent at the desired concentration, followed by stirring at 80 °C for 15 minutes. The hot and hazy samples were subsequently vortexed for 15 seconds and this procedure was repeated again if the sample still looked hazy. All samples were left to equilibrate at room temperature overnight and samples in D2O were viscous the next day. First a background of the appropriate solvent was measured. All spectra were measured at room temperature from 400 cm -1 to 4000 cm -1 , averaged over 64 scans.
Cryogenic transmission electron microscopy (cryoTEM) images were made of vitrified samples with a concentration of 500 μM. Vitrified films were prepared in a 'Vitrobot' instrument (FEI Vitrobot TM Mark IV, FEI Company) at 22 °C and at a relative humidity of 100%. In the preparation chamber of the 'Vitrobot', 3 μL samples were applied on Quantifoil grids (R 2/2, Quantifoil Micro Tools GmbH), which were surface plasma treated just prior to use (Cressington 208 carbon coater operating at 5 mA for 40 s). Excess sample was removed by blotting using filter paper for 3 s with a blotting force of -1, and the thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. Vitrified films were transferred into the vacuum of a CryoTITAN equipped with a field emission gun that was operated at 300 kV, a post-column Gatan energy filter, and a 2048 x 2048 Gatan CCD camera. Virtrified films were observed in the CryoTITAN microscope at temperatures below -170 °C. Micrographs were taken at low dose conditions, starting at a magnification of 6500 with a defocus setting of -40 µm or at a magnification of 24000 with a defocus setting of -10 µm.
2D image classification and averaging was performed in Berlin on samples with a concentration of 500 µM. The method is based on previously reported procedures. 1 Vitrified films were prepared in a 'Vitrobot' instrument (Vitrobot TM Mark IV, Thermo Fisher Scientific) at 22 °C and at a relative humidity of 100%. In the preparation chamber of the 'Vitrobot', 4 μL samples were applied on Quantifoil grids (R 1/4) which were surface plasma treated with a BALTEC MED 020. Excess sample was removed by blotting using filter paper for 3.5 s, a drain time of 1.0 s, a wait time of 1.0 s and with a blotting force of -13. The thin film thus formed was plunged (acceleration about 3 g) into liquid ethane just above its freezing point. Vitrified films were imaged with a Talos Arctica TM TEM (Thermo Fisher Scientific) at 200kV accelerating voltage at temperatures below -170 °C. Image recording was done using a Falcon3EC direct electron detector (Thermo Fisher Scientific). Micrographs were taken at low dose conditions, starting at a magnification of 28000x with a defocus setting around -5 µm. From cryo-TEM images, individual motifs (128 x 128 pixels, 0.373 nm/pixel) were extracted using the EMAN tool boxer. 2 Utilizing the image processing software package IMAGIC-5, 3 79 images were aligned with respect to one or multiple reference images, using cross-correlation techniques. The images were furthermore band-pass filtered to exclude low and high spatial frequencies, thus reducing unspecific noise. Subsequently, a mask image was generated, isolating the area of interest in the images. This mask was applied to all images and the multivariate statistical analysis was computed to confirm the absence of other morphologies or artefacts.
Hydrogen deuterium exchange experiments with electrospray ionization were carried out using a Xevo TM G2 QTof mass spectrometer (Waters) with a capillary voltage of 2.7 kV, a cone voltage of 80 V and an extraction cone voltage of 4.0 V. The source temperature was set at 100 °C, the desolvation temperature at 400 °C, and the cone gas flow at 10 L/h and the desolvation gas flow at 500 L/h. The sample solutions subjected to H/D exchange were introduced into the mass spectrometer using a Harvard syringe pump (11 Plus, Harvard Apparatus) at a flow rate of 50 µL/min. The signal was left to equilibrate for 1 measurement before starting the measurement and each measurement was averaged over 1 minute to account for instabilities in the signal. Spectra were recorded in centroid mode and the intensity of the peaks is used for the calculations as described in section 6. Before each measurement, the system was calibrated with a 0.05% H3PO4 solution in 1:1 H2O:ACN. Isotope patterns for calculation were determined with IsoPro software.

BTA-(S)-D-C12-EG4
was synthesized based on previously reported literature procedures 4 (see Scheme S1). A new route towards aldehyde 4 was used to prevent the formation of side products. A deuterium isotope was stereoselectively introduced at the α-position of each dodecyl chain with the alcohol dehydrogenase of Thermoanaerobacter sp. (ADH-T, (S)-selective for reduction of deuterated aldehydes with isopropanol, 331 U/mL) with nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and isopropanol-d8 as deuterium source. 5 The formed chiral (R)-alcohol is subsequently converted to an (S)-amine and coupled to the BTA core.

Assessing the enantioselective introduction of deuterium atoms
The stereoselectivity of chiral (R)-alcohol 5 was studied with (R)-α-methoxy-αtrifluoromethylphenylacetyl chloride (MTPA-Cl) derivatization of the alcohol (Scheme S2). 5 With this tool diastereomeric esters are formed which facilitates the determination of the ee of the chiral alcohol.  The OC-H proton resonates at 4.31 ppm in the 1 H NMR spectrum which corresponds to the (R)-alcohol as reported in literature. 5 No overlap of the (S)-alcohol was detected, indicating that the deuterium atom was introduced with an enantiomeric excess of > 95%. Based on literature reports we assume that this high stereoselectivity does not decrease upon conversion to the (S)-amine. 5 Zoom-in of the 1 H NMR spectrum of 10 to visualize the OC-H peaks.

Sample preparation for supramolecular polymers in water
Sample preparation of BTA-C12-EG4 and BTA-(S)-D-C12-EG4: the solid material was weighed into a glass vial equipped with a magnetic stirring bar. MQ-water was added to obtain the desired concentration. The sample was subsequently stirred at 80 °C for 15 minutes and the hot and hazy sample was vortexed immediately afterwards for 15 seconds. All samples were left to equilibrate at room temperature.
Sample preparation of mixtures: 2.5 mM stock solutions of the BTAs were prepared in ACN. Stock solutions were mixed in the desired ratio in a separate vial with magnetic stirring bar. ACN was evaporated with a stream of N2 (g) in samples. MQ-water was added to obtain the desired concentration. The sample was subsequently stirred at 80 °C for 15 minutes and the hot and hazy sample was vortexed immediately afterwards for 15 seconds. All samples were left to equilibrate at room temperature. in MeOD and D2O (c = 20 mg/mL, l = 0.05 mm, T = room temperature). The peak at 1647 cm -1 corresponds to the absence of intermolecular hydrogen bonds, whereas the peak at 1633 cm -1 corresponds to hydrogen bonded supramolecular polymers, thereby confirming the formation of intermolecular hydrogen bonds in aqueous solution. 7 The vibrations at 1708 cm -1 in MeOD 8 and 1697 cm -1 in D2O 9 originate from the C=O vibration of acetone which was used to clean the cuvette.   The lines represent a bi-exponential growth function added to guide the eye.